Drug Metabolism and Pharmacogenetics

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Drug Metabolism and Pharmacogenetics

• Brendan Stamper
• University of Washington
• Dept. of Medicinal Chemistry

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What is Medicinal Chemistry?

• Medicinal Chemistry is a scientific discipline
  involved with designing, synthesizing and
  developing pharmaceuticals suitable for
  therapeutic use
• Highly interdisciplinary science combining
  genetics, molecular biology, biochemistry,
  organic chemistry, pharmacology, toxicology

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Outline

• Background
• Drug Metabolism
• Pharmacogenetics
• Examples
• CYP2D6
• Codeine
• DDI Scenario (fluoxetine)
• ALDH2
• Ethanol
• DDI Scenario (acetaminophen)

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Basic Vocabulary

• Lipophilicity vs Hydrophilicity
• Lipophile Fat-lover
• Hydrophile Water-lover
• Xenobiotic a foreign chemical substance

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More Basic Vocabulary

• Metabolism chemical reactions that occur in
  living organisms
• Enzyme a biomolecule that catalyzes a chemical
  reaction

Inducer
Enzyme
Xenobiotic
Metabolite
Inhibitor
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Todays Focus

• We will focus on the enzymatic conversion of
  lipophilic xenobiotics to more water soluble
  metabolites . . .
• . . . and how these processes are influenced
  by genetic predisposition

WHY DO WE CARE?
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• Lipophilic xenobiotics can be potentially
  dangerous because they can easily permeate lipid
  cell membranes and accumulate within cells
• By converting lipophilic xenobiotics to
  hydrophilic metabolites we can facilitate
  elimination

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Todays Focus

• We will focus on the enzymatic conversion of
  lipophilic xenobiotics to more water soluble
  metabolites . . .
• . . . and how these processes are influenced
  by genetic predisposition

(i.e. Can we expect xenobiotic metabolism to be
consistent from person-to-person?)
NO
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Variability in Dose-Response

• If it were not for the great variability
  among individuals, medicine might as well be a
  science and not an art

William Osler 1849-1919
What factors are responsible for this
variability?
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Variability in Dose-Response
Genetics
Age
Race
Drug Response
Disease
Stress
Gender
Diet
Occupational Exposure
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Pharmacogenetics

• Definition The study of genetic variation that
  gives rise to variability in drug response
  (Optimize efficacy and limit toxicity)

Genetics
Age
Race
Drug Response
Disease
Stress
Gender
Diet
Occupational Exposure
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How do we predict optimal dose for most
efficacious response

• Step 1 Understand the mechanism of drug action
  at the molecular level
• Step 2 Understand how genetic variations affect
  drug action
• Step 3 Rational choice of drug and dosage

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DNA Is Like a Language

• DNA
• ATGC
• Codon
• Gene
• Chromosome
• Genome

• English
• Abcdef . . .
• Word
• Sentence
• Chapter
• Book

Like language, DNA changes over time
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Polymorphism

• Polymorphism Change in DNA sequence that occurs
  in more than 1 of the population
• Allele An alternative form of a gene (i.e. site
  of sequence variation)
• SNP (Single Nucleotide Polymorphism)
• Gene ATG-GGA-TGC-TAA met-gly-cys-STOP
• SNP ATG-GCA-TGC-TAA met-ala-cys-STOP
• Impact of new allele
• Alter protein function
• Alter protein structure or stability
• No consequence

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CYP2D6 and Codeine

• Example 1

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Codeine

• Analgesic
• Prodrug
• CYP2D6-mediated bioactivation critical for
  analgesic effect
• 200mg codeine is equivalent to 30mg morphine
  (10)

CYP2D6
More Lipophilic
More Hydrophilic
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CYP2D6

• Drug metabolizing enzyme
• Member of the P450 family (Cytochrome P450 2D6)
• Common substrates
• Beta-blockers (Metoprolol)
• SSRIs (Fluoxetine)
• Opiods (Codeine)
• SERMs (Tamoxifen)
• Highly polymorphic enzyme
• Over 100 reported

Rowland et al, JBC (2006) 2817614-7622
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Allele Kinetic Data
Kinetic plot of product formation versus
substrate concentration for metabolic turnover of
codeine catalyzed by highly purified recombinant
CYP2D6 isoforms in vitro.
Yu et al, JPET (2002) 3031291-1300
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CYP2D6 Genotypes

• Ultra Metabolizers
• Extensive Metabolizers
• Intermediate Metabolizers
• Poor Metabolizers

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Poor vs Extensive Metabolizers
Individual plasma concentration of codeine and
morphine in 14 extensive (filled) and 14 poor
(open) metabolizers after an oral dose of codeine.
Poulsen et al, Eur J Clin Pharmacol (1996)
51289-295
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How can we sort the population into the different
CYP2D6 metabolizing groups?
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Urinary Metabolic Ratio of CYP2D6 Substrate

• Dose patients with CYP2D6 substrate (codeine)
• Collect urine sample that contains substrate and
  metabolite
• Calculate ratio of substrate over metabolite

Substrate (codeine) Metabolite (morphine)
High (poor metabolizer) Low (extensitve
metabolizer)

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Urinary Metabolic Ratio of CYP2D6 Substrate
Intermediate Metabolizers
Roden et al, Ann Intern Med 2006 145749-757
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Outcomes of CYP2D6 Allelic Variations
No analgesic effect
Slight analgesic effect
Pain relief
Overdose effect
Null allele
Expected plasma concentration-time curve with
therepeutic window indicated by the boxed area
Decreased function allele
Fully functional allele
Zanger et al, Naunyn-Schiedebergs Arch Pharmacol
(2004) 369 23-37
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Codeine Pharmacogenetics
Green Intermediate and Extensive
Metabolizers Purple Ultra Metabolizers Orange
Poor Metabolizers
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Who are most affected by CYP2D6 polymorphisms?
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Ethnic Variation in CYP2D6 Mutation Frequencies
Variant Phenotype Caucasian Asian African Ethiopian/ Saudi
CYP2D62xN UM 1-5 0-2 2 10-16
CYP2D64 PM 12-21 1 2 1-4
CYP2D610 IM 1-2 51 6 3-9
CYP2D617 IM 0 ND 34 3-9
CYP450 allele nomenclature committee database
http//www.imm.ki.se/cypalleles
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DDI ScenarioCodeine and Fluoxetine

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Drug-Drug InteractionsCodeine Fluoxetine
CYP2D6
X
Codeine
Morphine
Fluoxetine (inhibitor)
Zanger et al, Naunyn-Schiedebergs Arch Pharmacol
(2004) 369 23-37
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Summary CYP2D6 Codeine

• Codeine is a prodrug (requires metabolism)
• CYP2D6 metabolizes codeine to morphine
• CYP2D6 is a highly polymorphic enzyme
• Populations can be separated into different
  metabolic sub-groups
• UMs Overdose analgesic effect
• EMs/IMs Predicted analgesic effect
• PMs No analgesic effect
• Urinary sampling can enable the pre-sorting of
  different metabolizers
• Co-treatment with fluoxetine EMs to PMs

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ALDH2 and Ethanol

• Example 2

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Ethanol

• Low dose Muscle relaxant, euphoria impaired
  judgment
• High dose CNS depressant, impaired sensory/motor
  function
• Toxic when BAC gt 400mg/dL (0.4)

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Ethanol Metabolism
ADH
ALDH
More Lipophilic
More Hydrophilic

• Alcohol to aldehyde to carboxylic acid
• Ethanol CNS depressant
• Acetaldehyde Vasodilator
• Flushing
• Hangover effects
• Acetic Acid Relatively harmless

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ALDH2

• Aldehyde dehydrogenase 2
• Mitochondrial enzyme
• Homotetramer
• Substrates aldehydes
• Cofactor NAD
• Catalyzes the oxidation of aldehydes
• Polymorphic enzyme

Larson et al, JBC (2005) 28030550-30556
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ALDH2 Genotypes

• ALDH21/1 Wild-type Homozygous
• ALDH21/2 Heterozygous
• ALDH22/2 Mutant Homozygous
• 2 allele E487K mutation

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ALDH Crystal Structure
2 Mutation E487K
Violet Blue NAD-bound ALDH21 Red NAD-bound
ALDH22
Larson et al, JBC (2005) 28030550-30556
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ALDH2 Activity Among Differing Genotypes
MALD
AALD
BALD
Comparison of substrate specific activities of
human liver ALDH2 derived from three ALDH
genotypes
PALD
Kitagawa et al, FEBS Letters (2000) 476306-311
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ALDH2 Influence on AALD Blood Levels Following
Ethanol Ingestion
Ginsberg et al, Reg Toxicol Pharmacol (2002) 36,
297-309
ADH
ALDH21/1
ALDH21/2
ALDH22/2
X
No Flushing No Hangover Effects
Some Flushing Some Hangover Effects
Flushing Hangover Effects
X
X
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Who is Affected?
ALDH2 polymorphism by ethnic group
Ginsberg et al, Reg Toxicol Pharmacol (2002) 36,
297-309
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How do you treat an acetaldehyde overdose?
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DDI Scenario(Ethanol and Acetaminophen)

Analgesic/Antpyretic
CNS Depressant
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Drug-Drug InteractionEthanol and Acetaminophen
Glutathione adducts (Detoxification)
X
CYP2E1
Protein adducts (Toxicity)
Ethanol (inducer)
Cell Death
NAPQI
Liver Damage
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Toxic Scenario

1. Chronic alcohol abuser induces CYP2E1
2. Activated CYP2E1 forms more toxic metabolite
   (NAPQI)
3. Increased levels of NAPQI can lead to glutathione
   depletion
4. Increased protein adduct formation leading to
   cell death and liver damage

If a you are a chronic alcohol abuser, use
ibuprofen instead of acetaminophen to treat your
hangover.
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Summary ALDH2 Ethanol

• Ethanol metabolism occurs in two steps
• ADH Ethanol to acetaldehyde (toxic metabolite)
• ALDH2 Acetaldehyde to acetic acid
  (detoxification step)
• Three common ALDH2 genotypes
• ALDH21/1 No flushing or hangover effects
• ALDH21/2 Some flushing and hangover effects
• ALDH22/2 Flushing and hangover effects
• ALDH22 most prevalent in Asian populations
• Chronic alcohol abusers should not take
  acetaminophen to treat their hangovers

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Things to Think About

• Drug metabolizing enzymes tend to metabolize
  lipophilic compounds into hydrophilic compounds
  (absorption to excretion)
• Your genotype impacts how you metabolize drugs
• Pharmacogenetics can be used to optimize therapy
• Science is interdisciplinary

Genetics - Molecular Biology - Biochemistry -
Organic Chemistry - Pharmacology - Toxicology
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Thank you!